Cannabinoids, Inner Ear, Hearing, and Tinnitus: A Neuroimmunological Perspective This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). Cannabinoid CB 1 Receptor Agonists Do Not Decrease, but may Increase Acoustic Trauma-Induced Tinnitus in Rats This is an open-access article distributed under the terms of the Creative Commons
Cannabinoids, Inner Ear, Hearing, and Tinnitus: A Neuroimmunological Perspective
This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
Cannabis has been used for centuries for recreational and therapeutic purposes. Whereas, the recreative uses are based on the psychotropic effect of some of its compounds, its therapeutic effects range over a wide spectrum of actions, most of which target the brain or the immune system. Several studies have found cannabinoid receptors in the auditory system, both at peripheral and central levels, thus raising the interest in cannabinoid signaling in hearing, and especially in tinnitus, which is affected also by anxiety, memory, and attention circuits where cannabinoid effects are well described. Available studies on animal models of tinnitus suggest that cannabinoids are not likely to be helpful in tinnitus treatment and could even be harmful. However, the pharmacology of cannabinoids is very complex, and most studies focused on neural CB1R-based responses. Cannabinoid effects on the immune system (where CB2Rs predominate) are increasingly recognized as essential in understanding nervous system pathological responses, and data on immune cannabinoid targets have emerged in the auditory system as well. In addition, nonclassical cannabinoid targets (such as TRP channels) appear to play an important role in the auditory system as well. This review will focus on neuroimmunological mechanisms for cannabinoid effects and their possible use as protective and therapeutic agents in the ear and auditory system, especially in tinnitus.
Endocannabinoids (ECs; Figure 1 ) are a class of ubiquitous endogenous lipids regulating essential processes ranging from energy balance, to pain, to motor control, and involved in pathologies as diverse as (among others) schizophrenia, glaucoma, multiple sclerosis, and obesity (20). In the CNS, ECs influence synaptic plasticity (21, 22), modulate neuroinflammation (23), and affect neurogenesis (24) and may also affect neuronal activity by binding to neurotransmitter receptors and ion channels (25). These cellular effects are reflected in the EC modulation of several brain functions, including fear and anxiety (26), or memory (27). Overall, the standard arrangement in the brain appears to be the presence of multiple EC pathways affecting the same circuits, often with different or even opposing effect.
EC and their effects. (A): Principal EC targets in neural and immune systems, and in the cochlea. (a) In most brain areas, 2-AG (purple) is synthetized by DAGL-α in neuronal dendrites and somata and catabolized by MAGL in presynaptic terminals, where CB1R (red) are also present. 2-AG is produced postsynaptically in a Ca 2+ -dependent way upon activation of metabotropic receptors (blue) and inactivated presynaptically near its target (1). For AEA (yellow), on the other hand, the biosynthetic enzyme NAPE-PLD is both pre- and postsynaptic, and the catabolic enzyme FAAH-1 is predominantly postsynaptic (1). Astrocytes are also involved in synaptic effects through an EC modulation of gliotransmission, and in addition EC effects on astrocyte mitochondria contribute to neuronal metabolism regulation. (b) In the cochlea, CB2R (green) are found in the organ of Corti (OC), basal cells of the stria vascularis (SV) and spiral ganglion (SG), whereas TRPV1 channels (blue) are found in the organ of Corti and marginal cells of the stria vascularis. (c) During neuroinflammation, several changes are seen in the EC system. The overall EC production increases. Activated microglia increases AEA production (yellow trapezoid) and CB2R expression (green). Astrocytes become activated and BBB is affected (both effects are counteracted by EC responses). (d) Cell activation may change CB2R expression as in macrophages (purple) (2) or CB2R subcellular localization as in B lymphocytes (blue) (3). (e) Anti-inflammatory EC responses in immune cells include the block of Th1 responses due to direct effects on T cells (green) and indirect effects on dendritic cells (yellow), apoptosis induction on several cell types, and the inhibition of proinflammatory cytokines and factors. (B): Principal EC receptors and their main intracellular pathways. Both 2-AG (purple trapezoid) and AEA (yellow trapezoid) act on CB1R and CB2R, which are class A GPCRs (4) coupled to Gi/o G-proteins (5, 6), reducing cAMP concentration (7). β-Arrestin binding (light red arrowheads) induces CBR internalization and switches receptor coupling, especially for CB1, also activating MAP kinase pathways (8–10) linked to nuclear effects (large light blue arrow). MAPK pathways are also activated through Gi βγ action (dotted line), both by CB1R and by CB2R (11). Functional CBRs have been also found in intracellular compartments such as the outer mitochondrial membrane (12), where they modulate cell energetic balance (13), and ROS production (14) or endoplasmic reticulum, where they may induce Gq-related Ca2+ release from intracellular stores (3). Moreover, ECs or related lipids activate nuclear PPARs (15). TRPV1 channels are often colocalized with CB1R and/or CB2R and activated by cytoplasmic ECs (16), increasing cytosolic Ca2+. Finally, orphan receptors may activate other pathways, e.g., GPR55 is linked to Gq-PLC and therefore contributes to cytosolic Ca2+ increase. Most cells only express a subset of these pathways. (C): Metabolism of 2-AG and anandamide (AEA). 2-AG (purple) is produced from DAG by DAG lipases (DAGLα and β). Biosynthesis of AEA (yellow) is more complex and may involve hydrolysis of NAPE membrane phospholipids by NAPE-PLD (which directly generates AEA) or sequential action of several enzymes (not shown), followed by a lyso-PLD (17). Although lipophilic, ECs have membrane transport mechanisms (EMT, light gray) (18). EC binding sites on CB1 (red) and CB2 (green) are extracellular, whereas on TRP channels (blue) the site is intracellular. Degradation of AEA is mainly due to FAAH, whereas 2-AG is primarily degraded by MAGL, and less by ABHD6 and ABHD12 (19). Created with Biorender.
In the immune system, ECs affect cell proliferation, migration, differentiation, cytokine production, and apoptosis (28). The two responses, immune and neural, interact in neuroinflammation, where ECs play major roles (29). Earlier studies suggested that neural effects of cannabinoids are mediated by CB1R activation (30) whereas immune effects are mediated by CB2R (31). However, it is important to stress that the separation of these biological actions is not as clear-cut as initially suggested (32), and other receptors can also be activated by ECs. The dizzying complexity of cannabinoid pharmacology (see Supplementary Tables 1, 2) requires a deep knowledge of the precise “fingerprint” of the molecular pathways affected by each compound, in each organ and each species, to dissect its effects.
The pharmacology of cannabinoids is very complex, for several reasons. First, more than one hundred phytocannabinoids (33) and at least 13 ECs (25) have been identified. Second, their lipidic nature makes unraveling their molecular interactions more difficult than for conventional transmitters (34). Third, CBRs are connected to several intracellular pathways ( Figure 1B ) and may produce different (even opposite) results depending on the particular ligand and its concentration (8, 9, 35) and on the cell repertoire of signal transduction molecules (36).
ECs are one of the four families of bioactive lipids (together with classical eicosanoids, SPMs, and lysoglycerophospholipids/sphingolipids), which are generated from PUFA precursors esterified into membrane lipids (37). The EC system includes CBRs, their endogenous ligands, and the proteins involved in EC formation, transport, and degradation.
The first discovered and best-characterized ECs are AEA and 2-AG (38–40). Several other EC lipid mediators (41–43) [and a family of EC peptides, named “pepcans” (44)] have also been described (see also Supplementary Table 1), but their endogenous functions have been less characterized.
ECs ( Figure 1C ) are produced “on demand” from membrane lipids by several Ca 2+ -dependent enzymes (17), and metabolic pathways for production, transport, and degradation differ for the various ECs, making it possible for cells to tailor their local EC repertoire (45) by regulating their local concentrations through modulation of their biosynthesis, transport, and degradation (46). Once released, ECs are rapidly deactivated by intracellular enzymes (47): AEA by FAAH1 and 2 [the latter not expressed in rodents (1)], and 2-AG mainly by MAGL, and less by ABHD6 and ABHD12 (19). In addition, ECs may be transformed in non-EC bioactive metabolites [e.g., by COX-2 (48)].
ECs ( Figure 1B ) bind and activate two specific G-protein-coupled cannabinoid receptors, CB1R and CB2R (49–51), plus additional targets (52), such as TRP channels (53), PPARs (54–57), and “orphan” G-protein coupled receptors such as GPR18 and GPR55 (58, 59). Most EC are able to activate both CB1R and CB2R, although with different potency and effects (60), whereas nonclassical targets may interact with limited EC subsets (Supplementary Table 1) and also non-EC ligands. A clear example is TRPV1, which is activated by AEA binding to a cytoplasmic site (16) but is also sensitive to other stimuli such as heat, vanilloids, protons, N-acyl amides, and arachidonic acid derivatives (61).
CB1R and CB2R are class A (rhodopsin-like) GPCRs (4), and both couple to Gi/o G-proteins (5, 6), reducing cAMP concentration (7). However, the coupling between CBRs and biochemical pathways is complex and context-dependent. First, CB1Rs may form homo- or heterodimers with other GPCRs (62), such as (among others) CB2Rs (63), A2A adenosine receptors (64), D2 dopamine receptors (65), μ opioid receptors (66), and orexin-1 receptors (67), whereas CB2R may dimerize with the CXCR4 chemokine receptor (68) and GPR55 (69). The presence of CB1R/CB2R heteromers makes it impossible to clearly separate the biological responses of CB1R and CB2R in vivo.
Second, CBRs show dimerization- and agonist-biased response, due to conformation-dependent binding by β-arrestins (10). Besides blocking interactions with Gi/o proteins, β-arrestin effects include CBR internalization and ERK pathway/Gs protein activation (9), so that cAMP levels may increase instead of decreasing depending on CBR receptor bias. Receptor coupling flexibility appears more limited (although not absent) for CB2Rs, which mainly activate Gi proteins, whereas CB1Rs may couple to Go, Gs, Gq, and G12/13, thus activating a very diverse network of responses (8). Both receptors are in addition able to activate ER stress pathways linked to autophagy (70).
The β-arrestin-dependent internalization of plasma membrane CBRs is linked to receptor degradation (9); however, functional CBRs have been found in the outer mitochondrial membrane (12), and in the endoplasmic reticulum, endosomes, lysosomes, and nuclear membrane (3). Subcellular localization affect CB-related responses: mitochondrial localization allows CBRs to modulate cell energetic balance (13), and ROS production (14), whereas endolysosomal localization is correlated with inflammation and phagocytosis (71). Moreover, intracellular receptor sites will be inaccessible to membrane-impermeant cannabinoid agonists and antagonists.
Besides G-protein-coupled receptors, TRP nonselective cation channels are being increasingly recognized as an integral part of the EC system (ionotropic EC receptors): six of the 28 TRP channels are sensitive to cannabinoids (53). Among these, TRPV1 is the most studied, mainly due to its expression in nociceptors and role in pain-related processes: TRPV1 channels are colocalized with CB1R and/or CB2R in several types of cells, and TRPV1 block or desensitization underlies analgesia (72); the analgesic and antihyperalgesic effects of phytocannabinoids are, at least in part, mediated by this channel (53).
EC System in the Brain
CBRs are expressed in most tissues of the body (73) and are by far the most abundant type of G-protein-coupled receptors in the mammalian brain (74). CB1R is predominantly expressed in the CNS (75), at comparable levels as glutamate and GABA receptors (74, 76). On the other hand, CB2R was originally thought to be restricted to immune and hematopoietic cells (77, 78), but more precise localization tools have subsequently allowed to assess its expression in other systems, including the nervous system (79) and the inner ear (80). CB2R expression in the healthy brain is in fact hundreds of times less than for CB1R but is strongly upregulated under pathological conditions (81). Localization, splice variants, and physiology of CBRs appear to be highly species-dependent (73), thus complicating result comparisons between animal and human studies.
CB1R neuronal effects are well known and have been extensively covered in several exhaustive reviews (49, 82, 83). Glial responses are less completely characterized but appear important especially in the presence of neuroinflammation (84), where EC tone is elevated (85). Neuroinflammation is a protective brain defense response that can however degenerate into a chronical state involved in the pathophysiology of several neurological and psychiatric disorders (86).
In neurons ( Figure 1A a), the classical EC effect is retrograde inhibition mediated by presynaptic neuronal CB1Rs and postsynaptically produced 2-AG: CB1R activation inhibits the release of the presynaptic transmitter (22), causing short-term DSE on excitatory neurons, or DSI on inhibitory neurons (87). This mechanism has been dissected in the DCN molecular layer, where glutamatergic parallel fibers carrying non-auditory signals contact fusiform cells and glycinergic cartwheel cells (which in turn provide feedforward inhibition to fusiform cells) (88). Fusiform cell output is shaped by plasticity in the molecular layer circuits, which collectively generate “negative images” of expected sounds to be attenuated at fusiform apical dendrites (89). Plasticity changes in this circuit have been correlated with tinnitus onset (90, 91). Cartwheel cells release EC from their dendrites upon stimulation, thus inducing DSE at parallel fibers (92), whereas fusiform cells do not; therefore, activation of cartwheel cells depresses its parallel fiber input, gradually reducing their feedforward inhibition (93). In fusiform cells, ECs are involved in acetylcholine-induced plasticity changes at parallel fiber synapses (94) which have been correlated with tinnitus (95). Prolonged exposure to high doses of salicylate (a well-known tinnitus inducer) increases EC release in the DCN, thus changing molecular layer plasticity (96). Unfortunately, cannabinoid modulation of this circuit has not yielded effective tinnitus treatments [see discussion in (97)].
For AEA, on the other hand, the biosynthetic enzyme NAPE-PLD is both pre- and postsynaptic, and the catabolic enzyme FAAH-1 is predominantly postsynaptic (1). Postsynaptic production of AEA produces a “tonic” retrograde inhibition at some synapses, which is shut down by neuronal inactivity through upregulation of FAAH1 (98); presynaptic production feeds instead into an anterograde mechanism. In addition, in the hippocampus, NAPE-PLD is localized in intracellular membrane cisternae of axonal Ca 2+ stores (99) and AEA may act as an intracellular messenger by activating TRPV1 intracellular binding site.
Like neurons, glial cells can synthesize ECs in response to physiological or pathological stimuli (100, 101). In astrocytes, more than 70% of CB1Rs are found at perivascular endfeet, and EC activation has been found to modulate brain energy consumption (102) through EC effects on astrocyte mitochondria (103). At synapses, astrocytes express both DAGLα and MAGL and may display Ca 2+ -dependent EC release, which modulates synaptic response (104); conversely, astrocytic CB1R activation may induce Ca 2+ -dependent release of Glu (105), ATP, or D-serine (106) in response to synaptic EC. Astrocyte EC effects have been found to be involved in the regulation of sleep in the PPT (107) and in the regulation of circadian rhythms in the suprachiasmatic nucleus (108). These latter effects may be relevant for tinnitus given its association with sleep disturbances (109) and its circadian modulation (110).
Neuroinflammation is a brain reaction aimed at counteracting acute damage, restoring the homeostasis and limiting brain parenchyma injury, and includes microglial activation, reactive astrogliosis, production of inflammatory mediators, BBB breakdown, and subsequent brain infiltration of circulating immune cells (111). Neuroinflammation dysregulation may turn microglia and astrocytes in uncontrolled sources of inflammatory mediators, which may worsen damage progression.
A growing body of data suggest that EC are able to exert immunoregulatory and anti-inflammatory properties (112–114), by decreasing the production of NO, ROS/RNS, free radicals, and pro-inflammatory cytokines in activated glial cells, while switching microglia toward anti-inflammatory phenotypes (115–118). Remarkably, the increase in EC concentration and microglial CB receptors during neuroinflammation may yield a neuroprotective negative feedback mechanism aimed at limiting inflammatory responses.
The main brain source of ECs in neuroinflammatory conditions is microglia (119, 120), the resident immune cells of the CNS (121–123). Consistently with its immune role and nature, microglia express DAGL-β and (mainly) ABHD12 instead of the neuronal DAGL-α and MAGL (124), and while CB1Rs are expressed at low levels and mostly located intracellularly (120), microglia is the main CB2R-expressing cell in the brain (125). Microglial CB2R expression may increase up to 100 fold upon inflammation or tissue injury (126), and microglial Ca 2+ increases [e.g., from P2X7 receptor activation (127)] and directly increases DAGL, thus increasing the production of 2-AG (128), which during neuroinflammation becomes 20-fold higher in microglia than in other brain cells (120). Mounting evidence suggests that the EC system might represent a promising tool to modify (micro)glial activity and profiles in order to achieve benefits for neuroinflammatory diseases (104). Indeed, CB2Rs can downregulate astrocyte and microglial cell overactivation during neuroinflammatory disorders, thus protecting them (129); selective depletion of MAGL in astrocytes attenuates LPS-induced neuroinflammation [(130), and CB2R upregulation and activation of EC signaling pathways have been associated with a restoration of tissue homeostasis in neuroinflammatory conditions (118, 131).
Brain CB2Rs have been less studied than CB1Rs (79), mainly due to the delay in the availability of sensitive genetic and molecular tools (126, 132, 133). In the CNS, CB2Rs are chiefly expressed on microglia (134, 135), and to some extent on astrocytes, oligodendrocytes, progenitor neural cells, and neurons (136–138); neuronal CB2R is mainly postsynaptic, differently from CB1R (137). In human, the brain only expresses one CB2R isoform (CB2RA) whereas a second one (CB2RB) is expressed in the immune system (139); rats express two additional isoforms (CB2RC and CB2RD) present neither in mice nor in humans (126), and their CB2R expression is lower and with a different distribution from mice (140). Lack of CB2R brain expression was incorrectly inferred by methods only evidencing non-brain isoforms or with insufficient sensitivity (126).
Microglial actions range from protection against damaging signals altering CNS homeostasis through phagocytosis, release of proinflammatory cytokines, and recruitment of circulating immune cells [reviewed in (141)], to controlling neuronal proliferation and differentiation [through selective neuronal phagocytosis and release of neurotrophic and neurotoxic factors reviewed in (142)], to modulating neuronal plasticity and memory [through neurotrophin release and selective synaptic pruning reviewed in (141, 142)]. In order to fulfill all these tasks, microglia are extremely plastic cells that readily change their phenotypes on demand; microglial phenotypes, previously crammed into an M1–M2 gradient to fit a classical macrophage activation model (143), are now recognized to be much more diverse (144) and influenced by the brain region (145), species (146), age (147), gender (148), and physiopathological state (149). In particular, neurodegenerative diseases appear to associate with specific microglial phenotypes which release pro-inflammatory mediators, as well as contributing to prolonged oxidative stress, leading to chronic neuroinflammation, which in turn drives neurodegeneration (141, 150, 151).
As regards hearing loss, which is a risk factor for tinnitus, chronic inflammation is seen as a major player in presbycusis [reviewed in (152)] and has been found to be associated with poorer hearing in a population-based cross-sectional study (153). Moreover, in mice, microglial ablation and TNF-alpha antagonism (154) both decrease tinnitus signs, and TNF-alpha KO mice are resilient to noise trauma-induced tinnitus (154). In human, gene polymorphisms in both TNF-alpha (155) and IL-6 (156) have been found to increase tinnitus risk in an elderly population with a history of occupational noise exposure. Neuroinflammation (and its dysregulation) appears therefore as a promising candidate mechanism for tinnitus susceptibility, and its modulation by cannabinoids may provide novel therapeutic targets. A caveat regarding neuroinflammation as a target is the complexity emerging from single-cell studies (157), which could underlie a heterogeneity similar to that observed in most multifactorial inflammatory disorders [e.g., rheumatoid arthritis (158), Menière’s disease (159), and IBD (160)].
Besides neurons and glial cells, neuroinflammation involves cells of the immune system, where EC cellular mechanisms differ from neuronal ones. Cannabinoid immunomodulatory effects are complex but appear to be largely mediated through CB2Rs, whose expression on immune cells is usually higher than that of CB1R (161, 162). Moreover, nonclassical cannabinoid targets such as TRP channels (53) and PPARs (15) are well-known as key regulators of the immune response (163–165). It is interesting that EC responses in the cochlea (see below) appear more similar to those observed in the immune system than in the nervous system.
In human immune cells, CB2R is expressed most in B cells, followed by NK cells, monocytes, neutrophils, and finally T cells (134, 166, 167). Peripheral blood T cells, monocytes, and dendritic cells only express intracellular CB2R (168), whereas naïve peripheral blood B cells also express these receptors on the cell surface and lose it upon activation (169). Intracellular CB2Rs in immune cells have been associated with Ca 2+ release from stores (3).
CB2R activation in immune cells regulates all three major MAPKs (12) and decreases DNA binding for various nuclear factors (170), which results in the downregulation of critical immunoregulatory genes including IL-2 (171, 172). Overall, these major signaling networks play important roles in CB2R-mediated effects on immune cell functions including migration, proliferation, differentiation, apoptosis, and cytokine production (28). Generally, effects of the EC system on immune cells appear directed toward an anti-inflammatory action, although the context-dependent action of cannabinoids may support different responses in different cell types and states (62, 173–176).
As regards neuroinflammatory responses, a major player is the Toll-like receptor (TLR) system (177). TLRs are able to recognize pathogen-associated and damage-associated molecular patterns (PAMPs and DAMPs), and several of their effects appear to be counteracted by ECs [especially through CB2R-related mechanisms (11)]. Since cochlear damage has also been found to induce TLR4-responses (178), similar protective effects could be expected on the cochlea.
Cannabinoids and Tinnitus
Cannabinoids have been considered as potential treatment for tinnitus percept and/or distress, and with the legalization of light cannabis (L.242/2016 as regards Italy), several tinnitus sufferers are turning to it as a possible DIY remedy. Interest in cannabinoids as possible treatment for tinnitus has been motivated by several reasons. Early models of tinnitus stressed its similarities with neuropathic pain (179) and with epilepsy (180), both of which can be modulated by cannabinoids (181, 182).
The association between tinnitus and marijuana use in humans has been studied with contrasting results. In one study on health problems related to illicit drug use from the NSDUH database (n = 29,195) (183), tinnitus did not show any association to marijuana use (whereas an association was found with hallucinogens and inhalants); in a second, cross-sectional study on the NHANES database (n = 2,705) (184), a correlation was found between tinnitus and cannabis use, although not between cannabis use frequency and tinnitus severity, and the authors concluded that it was not possible to differentiate between causal association (cannabis use increases tinnitus prevalence), reverse causal association (tinnitus sufferers use more cannabis than non-sufferers), and association due to external common cause (i.e., anxiety, which increases both tinnitus risk and cannabis use).
Animal studies [reviewed in (97)] suggest that cannabinoids do not reduce, and may even favor, tinnitus percept. Similarly, tinnitus in humans has been sporadically observed in association with abuse of synthetic cannabinoid mixtures (185, 186).
These seemingly contradictory results arise from two inherent complexities in the problem under study. First, the responses to cannabinoids (even for the same compound mixtures) strongly depend on drug formulation, administration route, and concentration. Second ( Figure 2 ), tinnitus can result from many different mechanisms which are often hard to identify.
Tinnitus-related EC targets are present in the cochlea and central auditory system but also in CNS circuits altered in tinnitus; moreover, ECs may target phenomena which are known to be associated with tinnitus risk (e.g., anxiety) even though precise cellular mechanisms are uncertain. In panel “Tinnitus network,” numbers indicate as follows: 1: parahippocampal cortex; 2: ventromedial prefrontal cortex; 3: cingulate cortex; 4: amygdala; 5: dorsolateral prefrontal cortex; 6: insula (from 273). In panel “Auditory CNS” numbers indicate as follow: 1: cochlear nuclei; 2: auditory pons and midbrain; 3: medial geniculate body; 4: auditory cortex. In panel “Cochlea,” numbers indicate as follows: 1: spiral ganglion; 2: organ of Corti; 3: stria vascularis; 4: cochlear macrophages. Created with Biorender.
As regards the first complexity, it is important to stress that isolated and characterized phytocannabinoids, present in Cannabis sativa L. and a few other plant species (187, 188), include about 120 molecules (189), the most studied of which are Δ9-THC, mainly responsible for cannabis psychoactive effects (55), and cannabidiol (CBD), the major non-psychotropic component (190).
After the explanation of the structure–activity relationships in the Δ9-THC series (191, 192), a large and heterogeneous array of cannabimimetic compounds (Supplementary Tables 1, 2) have been synthesized (193, 194) including cannabinoid receptor agonists and antagonists (195), as well as drugs acting on EC metabolism (18, 196). Although, for several of them, dangerous health effects and strong potential for abuse and addiction greatly limit therapeutic use (197–199), several synthetic and phyto-cannabinoids are currently under clinical evaluation for different pathological conditions (see Table 1 ).
Major clinical trials based on pharmacological treatment targeting the endocannabinoid system (updated to July 21, 2020).
|Drug||Pharmacology||Phase||Conditions||Completion date (*estimated date for ongoing studies)||National clinical trials (NCT) number|
|AZD1940||CB1/CB2 non-selective agonist||1
| > NCT00689780,
|Org 28611||CB1/CB2 non-selective agonist||2||Pain||August 2007||> NCT00782951|
|SAB378||CB1/CB2 non-selective agonist||2||Pain||January 2010||> NCT00723918|
|APD-371||CB2 selective agonist||2
|Abdominal Pain, Crohn disease
| > NCT03155945
|GW-842,166X||CB2 selective agonist||1
| > NCT00511524
|Lenabasum||CB2 selective agonist||2
|Chronic inflammation||*August 2020
| > NCT03451045
|Cannabidiol (Epidiolex)||See cannabidiol section for details||1,2
| > NCT02324673
| > NCT04193631
| > NCT02548559
| > NCT04086342
| > NCT02929264
|ASP8477||FAAH inhibitor||2||Neuropathic pain||February 2015||> NCT02065349|
| > NCT02169973
| > NCT00836082
| > NCT00822744
|Neuropathic pain||July 2011
| > NCT01634529
Cannabinoid bioavailability varies significantly by their formulation and route of administration (200, 201) and is also affected by poorly controllable factors such as subjective inhalation characteristics (200, 202, 203) or hepatic first-pass metabolism (202, 204–206). This is particularly relevant because the expansion of legal use of cannabinoids, for medical and nonmedical purposes, has substantially increased the types of commercially available preparations (207).
Second, besides the intrinsic complexities of cannabinoid pharmacology, the main problem in attempting a pharmacological approach to tinnitus is the lack of a clear unifying causative hypothesis for this condition (208, 209). Current models of tinnitus include (1) a peripheral trigger [which is assumed to be reduced or altered cochlear input (210), even if transient (211) or “hidden” [but see (212)], or possibly a somatosensory trigger (210, 213)]; (2) an aberrant compensatory response in the brainstem [most likely more complex than a simple “gain increase” (91, 210, 214) as was initially postulated to compensate for reduced input (215)]; and (3) a reconfiguration of cortical pathways including auditory, attentional, salience-related, and emotion-processing networks [which is thought to be necessary for the tinnitus percept to emerge to consciousness (216, 217)]. Given the absence of a causative hypothesis for tinnitus, in this review we will consider cannabinoid effects linked to both tinnitus and its main risk factors such as hearing loss or anxiety.
In animal models, tinnitus may be induced by noise trauma or ototoxic drugs such as salicylate (218). In humans, tinnitus is associated with several risk factors such as hearing loss, head trauma, and endocrine and immune dysregulation (208); however, the association between risk factors and tinnitus is far from linear. For example, although hearing loss is the main risk factor for tinnitus, it is not always accompanied by it, and tinnitus may be present without hearing loss (208). Non-auditory brain circuits also play important roles: in particular, tinnitus shows comorbidity with anxiety and depression (208, 219) and chronic tinnitus is associated with changes in attentional, memory, and limbic circuits (220, 221). The hypothesis explaining the involvement of non-auditory circuits includes a misdirection of attention which stays anomalously focused on the tinnitus percept (216), the involvement of limbic circuits encoding distress (220, 221) and the “replaying” of phantom sounds from memory in the absence of real percepts (220, 221).
At each of the levels thought to be associated with tinnitus onset and chronicization there are both well-known and potential cannabinoid targets. EC mechanisms have been found in the auditory brainstem, and particularly in the DCN, which is thought to be a major site of tinnitus onset (91, 222, 223). These neuronal, CB1R-based mechanisms (see previous section for a discussion of DCN effects) were considered very promising for a cannabinoid-based tinnitus treatment; unfortunately, animal studies displayed no effects, or even tinnitus increase, upon treatment [see discussion in (97)].
In addition to these targets, however, several other EC mechanisms (mainly related to inflammation) are present in the auditory system and in other CNS regions important for tinnitus ( Figure 2 ). A protective EC mechanism is present in the cochlea (224, 225). Moreover, animal studies show inflammatory responses in the auditory cortex after tinnitus induction (154, 226), and inflammatory responses in the cochlea (154, 227, 228) and cochlear nuclei (229–232) after tinnitus-inducing treatments. Neuroinflammation may uncover novel EC-related therapeutic strategies, given the well-known anti-inflammatory effect of several cannabinoid drugs and pathways (see previous section).
In the auditory system, EC receptors and biosynthetic enzymes have been observed in several species and at several levels, and EC system modulation affects hearing at various levels. Moreover, several immune components and mechanisms known to be affected by EC modulation are also present in the auditory system, both peripheral and central. In the mammalian auditory system, EC system components or effects have been found in the cochlea (80), cochlear nuclei (93, 96, 233), MNTB (234), inferior colliculus (235, 236), and auditory cortex (237).
The hearing phenotypes of knockout mice for CB1R (238) and ABHD12 (239) have been characterized. In CB1R KO mice, high-frequency hearing is reduced but gap detection is improved, suggesting a change in auditory processing (238) or attentional modulation of perception, since in humans, chronic cannabis use is associated with attention-modulated deficit in PPI (240). Of relevance for tinnitus, CB1R KO mice also exhibit increased anxiety responses (241).
ABHD12 KO mice (239) and human ABHD12 nonsense mutations (242) display progressive hearing loss within PHARC syndrome. The absence of functional ABHD12 removes a catabolic pathway for 2-AG (see Figure 1 ); although the causative link between mutation and phenotype is still missing, a pro-inflammatory phenotype displaying microglial activation is observed (243), consistent with the expression of ABHD12 in both resting and activated microglia (242). Moreover, in the ABHD12 KO mouse the AA-related lipidome displays significant brain region-dependent changes (239) and macrophages increase LPS-induced cytokine production (244). On the other hand, the selective block of ABHD12 in adult mice does not induce hearing loss, suggesting developmental effects (245).
KO mice for CB2R (246) and other EC system components (239) are available, but their hearing has not been characterized; CB2R KO mice, on the other hand, display significant memory alterations (247).
In the cochlea, CB1R mRNA has been detected, and it decreases upon tinnitus-inducing salicylate treatment (248). However, the role of CB1Rs in the cochlea is still uncertain. On the other hand, CB2Rs have been found in rodent hair cells and pillar and Deiters’ cells, spiral ganglion and nerve, and stria vascularis basal cells (224), and their expression increases upon cisplatin administration (80). Cisplatin is known to be strongly ototoxic by inducing cochlear inflammation (249), and CB2R block or knockdown makes the cochlea more sensitive to cisplatin ototoxicity (224): moreover, treatment with the CB2R antagonist AM630 is in itself proinflammatory, suggesting the presence of a cytoprotective EC tone in the cochlea (224). In addition, EC protective role in the cochlea has been found to involve TRPV1 activation: TRPV1 channels are expressed in hair cells (especially toward the apical pole), pillar, and Deiters’ cells and in the marginal cells of the stria vascularis (250). The TRPV1 agonist capsaicin increases cochlear CB2R expression, and a CB2R-dependent mechanism induces the activation of STAT3; on the other hand, cisplatin induces the activation of proapoptotic factor STAT1 (225). The protective effect of capsaicin, which transiently induces STAT1 and TTS (225), is most likely due to the strong desensitization it induces on TRPV1 channels after a transient activation, similar to its effect in pain treatment (251).
CB1Rs are present in both ventral (VCN) and dorsal (DCN) cochlear nuclei of the rat; in the VCN, their role is unclear, but their expression decreases upon salicylate treatment, which induces tinnitus (233). In the DCN, salicylate does not change CB1R expression (233) but alters EC response on cartwheel cells (96). It is interesting to note the presence of CB1R (252) and CB2R (253) in the IV ventricle choroid plexus, especially because CB2R promote neural stem cell proliferation (254) and neurogenesis was observed in cochlear nuclei after deafferentation (255). In both man (256) and rat (257), there is a variable direct contact between the DCN surface and branches of the choroid plexus, where ECs released in the DCN molecular layer (92) could reach the plexus, possibly modulating its immune gate function (258).
As regards cortical effects important for tinnitus treatment, it is well known that anxiety (181) and attention (259) are strongly affected by cannabinoids. A point to be remembered is that, although cannabis use is associated with an acute anti-anxiety effect (260), chronic cannabis use may dramatically worsen anxiety (261, 262), thus exacerbating tinnitus severity. The anxiety-inducing effect of cannabis is correlated with its Δ9-THC content, and Δ9-THC alone may induce anxiety and paranoia (263); on the other hand, CBD appears to have opposite effects on anxiety (264) and is currently under clinical evaluation for the treatment of anxiety, psychosis, and posttraumatic stress disorder (190, 265, 266).
These data show that cannabinoid effects of possible relevance for tinnitus are very diverse and include anti-inflammatory, protective reactions and selective circuit modulation of “auditory context.” Since the anti-inflammatory route is starting to be explored as a possible therapeutic target in hearing loss (152) and tinnitus (154), interest has been raised for cannabinoids as a treatment option, and in particular for CBD, owing to its good toxicological profile in humans and lack of psychotropic effects. The recent availability of CBD preparations underlies anecdotal use reports by tinnitus patients; however, no controlled human studies have been performed yet.
Cannabidiol (CBD) is currently under clinical evaluation for the treatment of pain, anxiety, depression, sleep disorders, PTSD, headaches, and seizures (see Supplementary Table 1), all conditions which display analogies or associations with tinnitus (97, 179, 208). Despite such a wide spectrum of potentially interesting pharmacological properties, the practical effects of CBD on tinnitus are still underexplored.
Indeed, as of today the only study using CBD investigated the effects of a THC-CBD 1:1 mixture on noise trauma-induced tinnitus in the rat, showing no effects of daily treatment on tinnitus animals, and actually suggesting that cannabinoids might favor tinnitus onset, since treatment increased the fraction of animals showing tinnitus signs (267). These results agree with the effects of synthetic CB1R agonists (WIN55, 212-2, CP55,940, and ACEA) which have been tested in animal models of salicylate-induced tinnitus, with negative results [rat: (268); guinea pig: (269)]. It has to be remembered, however, that co-administered CBD and THC interact in a very complex way, and cannabinoid mixtures exert effects which may be very different from the simple combination of the effects of each drug per se (270). One example is CB1R activation in the cerebral cortex and hippocampus, associated with effects on cognition and memory (271): in this model, CBD is able to counteract THC-induced memory impairment (272).
In general, the pharmacodynamic of CBD appears particularly complex, with over 65 identified molecular targets, and different mechanisms proposed to explain its actions (190, 273, 274). Here we summarize only the CBD targets which may bear relevance for tinnitus.
On CB1R/CB2R, CBD has a very low affinity (in the μM range) and shows little agonist activity; on the other hand, it seems to antagonize CB1/CB2 synthetic agonist action with KB values in the nM range (275). It has been suggested that CBD acts as negative allosteric modulator of CB1R and as antagonist/inverse agonist of CB2R (276); in addition, it may indirectly affect CBR function by inhibiting FAAH activity, thus increasing endogenous anandamide levels (277, 278). For example, CBD neuroprotective effect after cerebral hypoxia–ischemia in immature pigs involves CB2R activation (279) and may be therefore due to EC increase rather than to a direct receptor effect.
Besides these effects, CBD acts as antagonist/inverse agonist of GPCR3, GPCR6, GPCR18, and GPCR55 (33, 280) and modulates serotonergic transmission acting as an allosteric agonist of 5HT1A receptor, a partial agonist of 5HT2A, and an allosteric inhibitor of 5HT3A (281–283). CBD protective effects on a BBB permeability model (284) required PPARγ and 5HT1A and were independent of CBRs. Similarly, CBD anti-depressant and anxiolytic effects also appear independent from CB2R (285) and linked to 5HT1A activation.
In the μM range, CBD may also activate adenosine A1 (286) and A2A receptors (287), activate glycine α1 (288) and α3 receptors (289), inhibit α7 nicotinic acetylcholine receptors (290), and allosterically modulate μ and δ opioid receptors (half maximal inhibition was observed at ~10 μM) (291). As a caveat, since CBD concentrations > 20 μM are unlikely to be attained in vivo (292), not all the described CBD pharmacological activities are likely to be physiologically meaningful.
Modulation of α7 nAChRs may be relevant for tinnitus since these receptors are expressed in cortical and hippocampal neurons and affect cognition and memory [reviewed in (293)]; moreover, these receptors are also expressed in microglia (294) and macrophages (295) and are involved in the vagal-mediated cholinergic anti-inflammatory response signaling through the JAK2/STAT3 pathway, decreasing levels of pro-inflammatory cytokines, such as TNF-α, IL-1β, and IL-6 and increasing levels of anti-inflammatory cytokines such as IL-10 (295–298).
Finally, CBD may affect several ion channels including voltage-dependent Na channels (299), T-type Ca channels (300), and TRPV1 and TRPV2 channels (301). In particular, CBD can act on TRPV-1, exhibiting an action similar to capsaicin, both in vitro (302) and in an animal model of acute inflammation (303). This is relevant since capsaicin is able to exert protective effects on cochlear inflammatory damage (225), and therefore, CBD may exert similar otoprotective actions.
Cannabinoids are involved in neural processing in the healthy auditory system, in protective reaction to auditory damage, and in most non-auditory circuits known to be associated with tinnitus.
Given the availability of a large number of drugs with a wide spectrum of different effects on the EC system, it appears possible that some of them may reduce tinnitus percept or risk factors rather than increase them, similar to what is seen, e.g., for anxiety (where EC-targeting drugs may either worsen or ameliorate it).
EC modulation of neuroinflammatory responses in the auditory system, in particular by CBD, which is neuroprotective, is anti-inflammatory, undergoes clinical trial as an anxiolytic, and acts on pathways involved in cochlear damage protection, may represent a novel pharmacological approach to hearing loss and tinnitus, although more data are necessary (especially on humans) to assess the therapeutic value of this or other EC drugs.
All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication. PP, RP, and PE contributed auditory expertise. AMT, FM, and MC neuroimmunological expertise. PE and CB pharmacological and clinical expertise.
Conflict of Interest
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
|ABHD4, ABHD6, ABHD12||αβ-hydrolase domain 4,6,12|
|BDNF||brain-derived nerve factor|
|BNST||bed nucleus of the stria terminalis|
|CBR; CB1R; CB2R||cannabinoid receptor, type 1, type 2|
|COX-2||cyclooxygenase type 2|
|CREB||cAMP response element binding protein|
|DCN||dorsal cochlear nucleus|
|DSE||Depolarization-induced suppression of excitation|
|DSI||Depolarization-induced suppression of inhibition|
|EMT||endocannabinoid membrane transporter|
|ERK||Extracellular signal-Regulated Kinase|
|FAAH||fatty acid amide hydrolase|
|PHARC||Polyneuropathy, Hearing loss, Ataxia, Retinitis pigmentosa, and Cataracts|
|PPAR||Peroxisome proliferator-activated receptors|
|PUFA||polyunsaturated fatty acids|
|SPM||specialized pro-resolving mediators|
|TRP||Transient receptor potential|
|VCN||ventral cochlear nucleus.|
Funding. This work was supported by AIT ONLUS donations to PP and FRG2019 grant to CB.
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Cannabinoid CB1 Receptor Agonists Do Not Decrease, but may Increase Acoustic Trauma-Induced Tinnitus in Rats
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Tinnitus has been suggested to arise from neuronal hyperactivity in auditory areas of the brain, and anti-epileptic drugs are sometimes used to provide relief from tinnitus. Recently, the anti-epileptic properties of the cannabinoid drugs have gained increasing interest; however, the use of cannabinoids as a form of treatment for tinnitus is controversial. In this study, we tested whether a combination of delta-9-tetrahydrocannabinol (delta-9-THC) and cannabidiol (CBD), delivered in a 1:1 ratio, could affect tinnitus perception in a rat model of acoustic trauma-induced tinnitus. Following sham treatment or acoustic trauma, the animals were divided into the following groups: (1) sham (i.e., no acoustic trauma) with vehicle treatment; (2) sham with drug treatment (i.e., delta-9-THC + CBD); (3) acoustic trauma-exposed exhibiting tinnitus, with drug treatment; and (4) acoustic trauma-exposed exhibiting no tinnitus, with drug treatment. The animals received either the vehicle or the cannabinoid drugs every day, 30 min before the tinnitus behavioral testing. Acoustic trauma caused a significant increase in the auditory brainstem response (ABR) thresholds in the exposed animals, indicating hearing loss; however, there was a partial recovery over 6 months. Acoustic trauma did not always result in tinnitus; however, among those that did exhibit tinnitus, some of them had tinnitus at multiple frequencies while others had it only at a single frequency. The cannabinoids significantly increased the number of tinnitus animals in the exposed-tinnitus group, but not in the sham group. The results suggest that cannabinoids may promote the development of tinnitus, especially when there is pre-existing hearing damage.
Tinnitus is the perception and conscious awareness of sound that is not physically present. These phantom sounds can be ringing or buzzing noises or sometimes hissing, grinding, or roaring. Many people experience tinnitus transiently at some time in their life, but for chronic tinnitus sufferers, the condition can be frustrating and debilitating. In severe cases, it can be extremely disturbing, and even lead to suicide (1). Tinnitus affects 25% of the American population at some stage in their life, with 8% of people experiencing persistent or chronic tinnitus (1). While the prevalence of chronic tinnitus normally increases with age, it is alarming that an increasing number of adolescents and young adults are experiencing it due to risky music-listening behaviors, such as prolonged exposure to high-volume music by using portable music players, or going to excessively loud nightclubs or attending pop/rock concerts (2).
Tinnitus can be caused by exposure to loud noise, as well as head and neck injuries; it can also develop as a result of inner ear infection, drug toxicity (e.g., aminoglycoside antibiotics), or as a result of aging (3, 4). Although the mechanisms underlying tinnitus are still not fully understood, the most likely cause of tinnitus is changes in neural activity in the brain, which is supported by both animal and human studies. In animals and humans with tinnitus, neurons in multiple areas of the brain become more active and more neurons fire at the same time in order to compensate for the hearing loss due to damage to the cochlear hair cells (5). Based on the idea that tinnitus is generated by neuronal hyperactivity in the brain, non-benzodiazepine anti-epileptic drugs, such as carbamazepine, are often prescribed [see Ref. (6, 7) for reviews]. However, the preclinical evidence supporting the use of such drugs is limited and contradictory, and the few clinical trials that have been conducted have yielded inconsistent results [see Ref. (4, 6–8) for reviews]. There is also evidence that cannabinoids can suppress epileptiform and seizure activity in animals (9–11). However, there has been no controlled study in humans of the effects of Cannabis or cannabinoids on tinnitus itself.
One problem is that Cannabis contains over 400 different chemicals, with 66 cannabinoid chemicals unique to the genus. Studies in neuropharmacology have tended to focus on the key psychoactive ingredient, delta-9-tetrahydrocannabinol (delta-9-THC); however, there are many other cannabinoids in Cannabis such as cannabinol and cannabidiol (CBD), and it is not always obvious which cannabinoid is exerting the observed effects. In addition to synthetic cannabinoid receptor agonists, such as dronabinol and nabilone, which are used clinically for the treatment of nausea, vomiting, and wasting, natural Cannabis extracts such as a 1:1 ratio of delta-9-THC and CBD (Sativex™), are used for the treatment of spasticity and chronic pain in multiple sclerosis (12).
There are two classes of cannabinoid receptors, the CB1 and CB2 receptors. The general consensus is that CB1 receptors are expressed mainly in the CNS, while the CB2 receptors are localized mainly to the immune system, peripheral nervous system, testes, and retina [see Ref. (13) for a review]. The presynaptic localization of many CB1 receptors and their inhibition of calcium influx at presynaptic terminals may be the basis for any anticonvulsant effects, depending on the neurotransmitter being released. Both Zheng et al. (14) and Tzounopoulos et al. (15) quantified CB1 receptor expression in the cochlear nuclei. Tzounopoulos et al. (15) observed CB1 receptors in the dorsal cochlear nucleus (DCN) at the parallel fiber/cartwheel cell, parallel fiber/fusiform cell synapses, and on the dendritic spines of cartwheel cells, using electron microscopy. Furthermore, Zhao et al. (16) demonstrated that both fusiform and cartwheel cells expressed diacylglycerol (DAG) α and β, the two enzymes necessary for the production of the endocannabinoid, 2-arachidonyl glycerol (2-AG). Therefore, there is substantial evidence for an endocannabinoid system within the DCN, which may be important for the development of tinnitus.
Only two studies to date have investigated the relationship between CB1 receptors in the CN and tinnitus. Zheng et al. (14) studied the expression of CB1 receptors in the DCN and ventral cochlear nucleus (VCN) of rats in which tinnitus had been induced using salicylate injections. They found a significant decrease in the number of neurons expressing CB1 receptors in the VCN compared to control animals. In the only animal study of the effects of cannabinoids on tinnitus itself, Zheng et al. (17) investigated the effects of two CB1 receptor agonists, WIN55,212-2 and CP-55940, on tinnitus induced by salicylate injections in rats. Neither WIN55,212-2 nor CP55,940 significantly reduced the conditioned behavior associated with tinnitus perception. However, 3 mg/kg WIN55,212-2 and 0.3 mg/kg CP-55940 did significantly increase this behavior in normal control animals, suggesting that these cannabinoids might induce tinnitus-related behavior.
Given the lack of evidence relating to the effects of Cannabis on tinnitus in humans and the recent data supporting the existence of an endocannabinoid system in the cochlear nucleus, the aim of this study was to further investigate the effects of cannabinoid drugs on acoustic trauma-induced tinnitus, using a 1:1 ratio of delta-9-THC and CBD, which is equivalent to Sativex™ used in the treatment of spasticity and chronic pain in multiple sclerosis (12).
Materials and Methods
Fifty male Wistar rats (300–350 g at the beginning of the experiments) were used in this study. The animals were housed in groups of 2–3 per cage under a 12:12 h light:dark cycle at 22°C and were water deprived throughout the tinnitus behavioral testing. All procedures were approved by the University of Otago Committee on Ethics in the Care and Use of Laboratory Animals.
Delta-9-THC and CBD were purchased from THC Pharm GmbH (Frankfurt, Germany). The drugs were dissolved in Tween 80 and Ethanol (1:1) to make a 50 mg/ml stock solution of the mixture of delta-9-THC and CBD. A working solution containing 1 mg/ml of delta-9-THC and 1 mg/ml of CBD was made freshly every day by diluting the stock solution with saline. This 1:1 ratio of delta-9-THC and CBD was designed to approximate the cannabinoid drug, Sativex™, which is used in the treatment of spasticity and chronic pain in multiple sclerosis in humans (12). Multiple doses of this mixture were not tested simply due to the expense of the drugs.
The animals were randomly divided into sham (n = 20) and exposed (n = 30) groups and exposed to either the acoustic trauma or sham procedure. One month later, the animals were tested for the behavioral signs of tinnitus using a conditioned lick suppression paradigm. Following the confirmation of tinnitus, the acoustic trauma-exposed animals were further divided into exposed-tinnitus and exposed-no tinnitus groups. The effects of cannabinoids on tinnitus were investigated by administering either vehicle or delta-9-THC (1.5 mg/kg, s.c.) and CBD (1.5 mg/kg, s.c.) every day, 30 min before tinnitus testing, throughout the tinnitus testing period for a total of 27 days. These doses were the maximum doses that could be used without causing sedation in rats, during a pilot study. The animals were then given a 2-week washout period for the drugs to be eliminated before being tested again for the behavioral signs of tinnitus.
Acoustic trauma to induce tinnitus
The animals were exposed to unilateral acoustic trauma using the methods described in our previous publications (18–22). Briefly, the animals were anesthetized with a fentanyl (0.2 mg/kg, s.c.) and medetomidine hydrochloride (0.5 mg/kg, s.c.) mixture and placed inside a sound attenuation chamber. A 16 kHz pure tone with an intensity of 115 dB, generated by a NI 4461 Dynamic Signal Acquisition and Generation system (National Instruments New Zealand Ltd.), was delivered to one of the ears for 1 h through a closed field magnetic speaker with a tapered tip (Tucker-Davis Technologies). The unexposed ear was blocked with cone-shaped foam and taped against the foam surface inside the sound attenuation chamber. The sham animals received the same anesthetics and were kept under anesthesia for the same duration as the acoustic trauma animals, but without acoustic trauma exposure.
Hearing levels were measured using auditory brainstem response (ABR) thresholds in both the ears of exposed and sham animals before the acoustic trauma, in both the ears of the exposed animals immediately after the acoustic trauma, in the ipsilateral ear of all exposed animals and in both ears of selected sham animals at the conclusion of the study. Briefly, the animals were anesthetized as previously described and acoustic stimuli were presented directly to the entrance of the ear canal using the same set-up as for the acoustic trauma. Stainless steel needle electrodes were placed s.c. at the vertex and over the bullae with a reference electrode at the occiput. ABR thresholds were tested for tone bursts presented at a rate of 50/s. Tone bursts (2 ms rise/decay, 1 ms plateau) were presented in a decreasing intensity series, beginning with levels that elicited distinct evoked potentials. Hearing thresholds were indicated by the lowest intensity that produced visually distinct potentials, progressing in 20-, 10-, and 5-dB steps for 8, 16, 20, and 32 kHz stimuli (18–22).
Behavioral assessment of tinnitus
The presence of tinnitus was assessed using a conditioned lick suppression paradigm as described in our previous publications (18–22). Briefly, the animals were water deprived and allowed to drink inside an operant conditioning test chamber (ENV-007, Med Associates Inc.) by licking through a sipper tube. The number of licks was sensed by an infrared photobeam and recorded on a computer. The animal’s free-feeding weight was taken as a baseline and the body weight was monitored every day before the behavioral testing. If a rat made less than 1000 licks during any given session, extra water was provided for 30 min in its home cage after the testing session and if there was a weight loss of 10% of their baseline body weight, extra water was provided outside the testing period. This water deprivation schedule typically kept their body weight at 90–95% of their baseline body weight and motivated the rats to produce reliable licks (1500–3500 licks per session) during the tinnitus testing sessions.
The conditioned lick suppression paradigm consisted of 15 min of testing every day and the animals went through three phases: the acclimation phase, the Pavlovian conditioned suppression training phase, and the frequency discrimination phase. During the acclimation phase, a broadband noise (BBN, 60 dB SPL) was presented throughout the 15 min session except at 10 random intervals, at which point 15 s acoustic stimuli presentations were inserted. Two of the 10 presentations were always speaker off periods (i.e., silence) and the remaining 8 were either BBN, 20 kHz tones or 32 kHz tones at 4 different intensity levels (30, 40, 50, and 70 dB SPL for BBN; 70, 80, 90, and 100 dB SPL for 20 and 32 kHz) in a random order with each stimulus repeated twice within each session. The type of stimulus was varied randomly between sessions, but remained constant within a session, and the stimulus presentations did not occur within 1 min of one another, or within 1 min of the beginning or the end of the session. The animals had three sessions of acclimation for each type of stimulus.
Following acclimation, each animal received conditioned suppression training in which a 3 s foot shock (0.35 mA) was presented at the end of each speaker off (silence) period. Over a few sessions, the animals learned the association between the speaker off and the foot shock and reacted to the speaker off by stopping licking. The number of licks in the 15 s period preceding the stimulus presentation and the number of licks during the 15 s of the stimulus presentation were recorded. The lick suppression was measured by comparing the number of licks in these two periods, i.e., the suppression ratio (SR):
where A is the number of licks in the preceding period and B is the number of licks in the stimulus presentation period. If a rat did not make any licks during the 15 s period preceding the stimulus presentation, the corresponding SR for this particular period was excluded.
Once the lick suppression was established (SR < 0.2), the rats were subjected to the frequency discrimination test, during which the acoustic stimuli were presented in the same manner as in the acclimation and the suppression training and each stimulus was tested for 5–6 sessions, with one session per day. Foot shock was delivered only if the SR for the speaker off period was >0.2. During the drug treatment, we allowed nine sessions (three sessions each frequency) for the animals to establish new associations with changes in their tinnitus status if there were any and data collected from the first nine sessions of drug treatment were discarded. During the first few days, more foot shocks were triggered by the animals, which suggested the re-establishment of conditioned suppression. Furthermore, animals were tested every day for a further 18 sessions (six sessions for each stimulus) during the drug treatment period. This ensured that the animals had enough time to be reconditioned and to produce reliable responses.
If a rat did not have tinnitus, it would associate the silence period with the foot shock and the presentation of the stimuli had no meaning to it, therefore, its drinking activity would not be affected during the acoustic stimuli presentation periods. However, if a rat had tinnitus, it would hear its tinnitus during the silence period and associate its tinnitus, instead of the silence, with the foot shock. Therefore, a stimulus with sensory features resembling tinnitus during the testing session should act as a conditioned stimulus and produce greater suppression during the stimulus presentation period. Using this method, we have successfully induced and assessed tinnitus in rats in our laboratory and confirmed that the duration of tinnitus can last as long as 10 months after the acoustic trauma exposure, although the hearing loss is temporary (18–21).
Criteria to identify tinnitus animals
Following the first tinnitus test and before the drug treatment, the frequency discrimination curve from each of the acoustic trauma-exposed animals was constructed for BBN, 20 and 32 kHz, respectively, and compared with the mean frequency discrimination curve from the sham group. The exposed animals with lower SRs that were clearly separated from the sham animals at two or more intensity levels measured were considered to have tinnitus. This procedure inevitably meant that the sample sizes for the different groups were unequal, which is potentially a problem for the statistical analysis of repeated measures data, e.g., using analysis of variance (ANOVA). For this reason, we did not employ repeated measures ANOVAs but rather, a linear mixed model (LMM) analysis with a restricted maximum likelihood procedure, because it does not assume a balanced design and also addresses the correlation structure of the repeated measures data (see below) (23–26).
All data were tested for normality and homogeneity of variance, and a LMM analysis was undertaken using SPSS 22. Where these assumptions were violated, the data were square root transformed and re-tested. The SR data for tinnitus assessment were analyzed with a LMM analysis using a restricted maximum likelihood procedure (18–20, 23–25). LMM analyses were used in preference to repeated measures ANOVAs because of the problems caused by extensive autocorrelation in repeated measures data; LMM analyses model the covariance structure of the repeated measures data in order to address this problem (23–25). The data were analyzed with group (sham-vehicle, sham-drug, exposed-no tinnitus-drug, or exposed-tinnitus-drug) as a fixed factor and intensity as a repeated measure. Akaike’s Information Criterion (AIC) was used to determine the most appropriate covariance structure. Where appropriate, Bonferroni’s corrected post hoc tests were used to make pairwise comparisons. Results were considered significant if P ≤ 0.05. The number of animals with or without behavioral evidence of tinnitus was compared before, during, and after the drug administration using Chi-square tests.
In general, acoustic trauma resulted in a frequency-dependent increase in the ABR thresholds in the ipsilateral ear, which was similar for the tinnitus and no-tinnitus groups and which recovered partially over 6 months post-exposure. Acoustic trauma caused a significant increase in the ABR thresholds in the exposed animals as indicated by a significant group effect (F3, 202.630 = 2.874, P = 0.037) (Figures (Figures1A,B). 1 A,B). Post hoc tests revealed that there was no difference in the degree of ABR threshold elevation between the exposed-no tinnitus animals and the exposed-tinnitus animals (P = 1.000). The increase in the ABR thresholds was specifically in the ear ipsilateral to the acoustic trauma exposure and across all the frequencies tested, since there was a significant side effect (F1, 240.459 = 189.928, P = 0.0001) and a significant frequency effect (F3, 517.500 = 9.861, P = 0.0001). Moreover, the increase was also frequency-dependent as there were significant differences between all of the frequencies tested with larger increases at higher frequencies (8 vs 16 kHz, P = 0.29; 8 vs 20 kHz, P = 0.0001; 8 vs 32 kHz, P = 0.0001; 16 vs 20 kHz, P = 0.0001; 16 vs 32 kHz, P = 0.030; and 20 vs 32 kHz, P = 0.036). However, a significant side × frequency interaction indicated that the frequency-dependent increase in ABR threshold was specifically due to the ipsilateral ear (Figures (Figures1A,B, 1 A,B, middle panel). An overall significant time effect (F2, 268.563 = 245.389, P = 0.0001) and a side × time interaction (F2, 253.408 = 187.320, P = 0.0001) also confirmed an ipsilateral increase in ABR thresholds following acoustic trauma. Although there was a considerable recovery of the ABR thresholds at 6 months following acoustic trauma (Figures (Figures1A,B, 1 A,B, right panel), pairwise comparisons revealed a significant difference between the ABR thresholds before and immediately after acoustic trauma (P = 0.0001), immediately and at 6 months after acoustic trauma (P = 0.0001) as well as before and at 6 months after acoustic trauma (P = 0.0001).
ABR thresholds for the ipsilateral (A) and contralateral (B) ears of sham-vehicle, sham-acoustic, exposed-no tinnitus-drug, and exposed-tinnitus-drug animals pre-exposure, immediately post-exposure, and 6 months post-exposure, as a function of stimulus intensity in dB SPL and frequency in kHz. Data are presented as means ± 1 SE.
At 1 month following the acoustic trauma, the animals underwent behavioral testing for the presence of tinnitus. After the completion of the test, the sham animals were randomly divided into two groups, vehicle and drug groups, and two mean frequency discrimination curves were constructed. A frequency discrimination curve was constructed for each of the exposed animals and compared with the two sham mean discrimination curves. The frequency discrimination curve showed a general increase in SR value with the increase in testing stimulus intensity, which reflects the increase in the discriminative nature between the testing stimulus and the conditioned stimulus (e.g., silence), i.e., the louder the testing stimulus is, the easier it is able to be distinguished from silence, therefore, the less suppression and the higher the SR value. Exposed animals were selected to become part of the exposed-tinnitus-drug group if two or more points on their frequency discrimination curve were clearly below the mean sham discrimination curves. The rest of the exposed animals were grouped as an exposed-no tinnitus-drug group. Based on this criterion, 6 animals were considered to experience tinnitus for BBN stimuli, 8 for 20 kHz stimuli, and 10 for 32 kHz stimuli. Among these animals, some of them had tinnitus at multiple frequencies while others had it only at a single frequency. Therefore, there were a total of 14 rats considered to have tinnitus. When the mean frequency discrimination curves were compared between these four groups, there was a significant group effect for BBN (F3, 46.485 = 5.155, P = 0.004), 20 kHz (F2, 46.550 = 4.386, P = 0.008) and 32 kHz (F2, 46.592 = 9.660, P = 0.000) stimuli (Figure (Figure2, 2 , left panel). Post hoc tests revealed a significant difference between the exposed-tinnitus-drug group and exposed-no tinnitus-drug group for all three stimuli tested (BBN, P = 0.02; 20 kHz, P = 0.01; 32 kHz, P = 0.0001), between the exposed-tinnitus-drug group and sham-drug group for 20 kHz stimuli (P = 0.034) and between the exposed-tinnitus-drug group and sham-vehicle group for 32 kHz stimuli (P = 0.039).
Frequency discrimination curves for sham-vehicle (n = 10), sham-acoustic trauma (n = 10), exposed-no tinnitus-drug (n = 24, 22 and 20), and exposed-tinnitus-drug animals (n = 6, 8, and 10) in response to acoustic stimuli for BBN, 20 and 32 kHz tones before, during and after the drug administration. Data are presented as means ± 1 SE.
In order to test whether the combination of THC and CBD could affect the perception of tinnitus in rats, the drugs were injected every day, 30 min before the tinnitus behavioral testing. During the administration of THC and CBD, there was a noticeable number of animals from the exposed-no tinnitus-drug group exhibiting greater lick suppression behavior in reaction to the presentation of the stimuli (Figure (Figure2, 2 , middle panel). When the mean frequency discrimination curves from the four groups were compared, there was a significant group effect for 20 kHz stimuli (F3, 45.006 = 6.346, P = 0.001) and a significant group × intensity interaction for 32 kHz (F12, 71.752 = 1.902, P = 0.048), but there was no group effect for BBN. Post hoc tests revealed that when presented with 20 kHz tones, the mean frequency discrimination curve for the exposed-no tinnitus-drug group was significantly shifted downward and there was a significant difference between the exposed-no tinnitus-drug and sham-drug groups (P = 0.009). Moreover, the difference between the exposed-no tinnitus-drug and exposed-tinnitus-drug groups had disappeared (P = 1.000), which suggests that some animals from the exposed-no tinnitus-drug group had developed tinnitus while receiving THC and CBD. Although there was no significant group effect when 32 kHz stimuli were presented, the significant group × intensity interaction indicated that animals from different groups reacted differently to different intensities of the 32 kHz tones. A close inspection of the frequency discrimination curves (Figure (Figure2, 2 , middle panel, third row) revealed that both the exposed-no tinnitus-drug and exposed-tinnitus-drug groups produced more lick suppression when the 32 kHz tone was presented at 100 dB SPL.
To find out whether THC and CBD would have any long-lasting effects on the animals’ tinnitus-like behavior, the animals were given a 2-week washout period during which the drug administration was stopped and the animals had free access to water and food. The tinnitus testing resumed after the washout period and there was only a significant group effect for 32 kHz tones (F3, 48.581 = 3.870, P = 0.015), but not BBN or 20 kHz tones. This significant group effect was due to the difference between the exposed-tinnitus-drug and sham-drug groups (P = 0.011).
In addition, the proportion of acoustic trauma-exposed animals that had behavioral signs of tinnitus was compared before, during, and after the drug administration for BBN, 20 or 32 kHz stimuli presentations (Figure (Figure3). 3 ). Although more animals displayed behavioral evidence of tinnitus during the administration of THC and CBD for all three stimuli tested, a significant increase in the number of tinnitus animals was evident only for 20 kHz stimuli (χ 2 = 10.94, df = 2, P = 0.004). There was no difference in the number of tinnitus animals before and after the drug administration.
Number of tinnitus and no tinnitus animals following acoustic trauma before, during, and after the drug administration.
Our results showed that following acoustic trauma, only a proportion of animals developed tinnitus and the rest did not. However, the combination of THC and CBD reversibly increased the number of tinnitus animals in the exposed, but not the sham groups, which suggests that THC and CBD may promote the perception of tinnitus if there is pre-existing hearing damage.
It has been shown that not every animal exposed to acoustic trauma develops tinnitus, with the reported tinnitus-induction rate varying from 30 to 80% [see Ref. (27) for a review]. Whether an animal develops tinnitus or not seems to be not directly correlated with the degree of hearing loss either immediately after the acoustic trauma exposure or a few months later [see Ref. (27) for a review]. In the present study, all of our rats exhibited elevated ABR thresholds across a range of frequencies immediately after the acoustic trauma. It has been reported that exposure to loud tones at 10 kHz resulted in an immediate hearing loss across a wide range of frequencies both below and higher (i.e., 6–24 kHz) than the exposed frequency using compound action potential audiograms (28), which is in agreement with our observations. However, it is believed that the maximum hearing loss following exposure to loud tones usually occurred at half an octave above the exposed frequency (29). However, in the present study the maximum hearing loss was at 32 kHz, which is a full octave above the exposed frequency of 16 kHz. This might be due to the presence of harmonic distortion of the 16 kHz tone. However, this harmonic tone at 32 kHz was measured 30 dB below the 16 kHz tones, which should be less likely to cause greater hearing loss, although unexpected damage could still occur due to the increased susceptibility of hair cells in the higher frequency regions to free radicals (30). It might also be necessary to measure hearing loss at 24 kHz following exposure to 16 kHz tones in order to confirm whether a greater hearing loss would occur at half an octave above the exposed frequency. Nevertheless, the hearing loss recovered substantially at 6 months following exposure. Although the ABR thresholds in the exposed animals did not completely return to the pre-exposure level, they were not different from the sham animals tested in parallel. Therefore, the slightly elevated ABR thresholds at the end of the experiment might have been due to age-related changes, although it seems less likely to be the case given that the age of our rats (9–10 months old) was a few months younger than the age of the Wistar rats (12–14 months old) showing age-related hearing loss (31). Nevertheless, the fact that our exposed animals had similar degrees of acute hearing loss and recovery and only some of them developed tinnitus, suggests that tinnitus development might not be reflected by the gross changes in ABR thresholds. Schaette and McAlpine (32) reported that human tinnitus subjects could have a normal audiogram and a normal amplitude of the centrally generated ABR wave V, but a significantly reduced amplitude of the auditory nerve-generated ABR wave I, which suggests a “hidden hearing loss” in these tinnitus patients. In addition, the relationship between altered ABR waveforms and tinnitus has also been studied in animals models (33, 34), with an increase in early ABR wave amplitude up to N3 (33) and both increases (33) and decreases (34) in latencies reported. In this study, the ABR was not measured either at 1 month post-exposure or during the drug treatment, when changes in the animal’s tinnitus status occurred. Therefore, it is impossible to make valid correlations using the currently available ABR results. Future studies are needed to further analyze changes in the different components of the ABR waves in animals with tinnitus.
Among the animals that exhibited behavioral evidence of tinnitus, i.e., the downward shift of the frequency discrimination curve, tinnitus manifested at different frequencies, with some animals experiencing it at multiple frequencies and within the same animal, there were fluctuations of tinnitus-like behavior in response to specific frequencies at different time points following the acoustic trauma. These observations are generally in agreement with our previous publications and those of others (35–38). Following the administration of THC and CBD, exposed-no tinnitus-animals showed increased suppression during the drug treatment for the 20 kHz stimulus and to a lesser extent, for the 32 kHz stimulus and a separate analysis looking at the proportion of animals experiencing tinnitus showed that there were more tinnitus animals during the drug treatment in response to the 20 kHz stimulus. Taken together, the results indicated that more animals shift from no-tinnitus to tinnitus status in the exposed-no tinnitus group, which suggests that THC and CBD may promote or enhance the perception of tinnitus in animals. However, following a 2-week washout period, this effect disappeared for 20 kHz stimuli. In addition, animals in the sham-drug group showed an up-shift in the suppression curve for both 20 and 32 kHz stimuli. The explanation for these effects is unknown; however, because delta-9-THC has a long half-life and is sequestered in fat (39), it is possible that this is due to some kind of delayed therapeutic effect. Perhaps another washout test at a later time point could provide more conclusive results.
Due to the fact that the number of animals exhibiting behavioral signs of tinnitus was similar before and after drug treatment, but significantly increased during drug treatment, this increase in the number of tinnitus animals cannot be explained simply by tinnitus fluctuation. In addition, we also observed greater lick suppression in response to the BBN, which has been suggested to be due to hearing loss (37). In this study, this increase in suppression in response to the BBN was only evident at 1 month post-exposure, but not at later time points, which might reflect temporary hearing loss following acoustic trauma. However, the ABR was not measured at 1 month post-exposure in this study; therefore, a definitive conclusion could not be drawn.
It has been shown that acoustic trauma results in neuronal hyperactivity in different areas of the brain including the cochlear nucleus, the inferior colliculus, the medial geniculate body, and the auditory cortex (5, 40–44). The hyperactivity, at least in the DCN, has been attributed to a decrease of GABAergic inhibition (45) and the burst firing of the fusiform cells (46). One type of GABAergic interneuron in the DCN is the cartwheel cells, which strongly inhibit fusiform cells through feed-forward inhibition (47). Presynaptic CB1 receptors have been found at the terminals of parallel fibers synapsing with the cartwheel cells and activating the presynaptic CB1 receptors, as a result of either the sustained firing of cartwheel cells or the application of a CB1 receptor agonist, significantly reducing the synaptic strength (15, 48). Therefore, it is conceivable that the activation of CB1 receptors on presynaptic terminals resulted in a decrease in GABA release from the cartwheel cells, which in turn resulted in a reduction in inhibition of the fusiform cells. It is interesting that this effect was only observed in animals that had been exposed to acoustic trauma, but not in sham animals. It has been reported that somatosensory input transmitted by parallel fibers produced a suppression-dominant effect on auditory processes in normal animals, but this effect was shifted to enhancement in exposed-tinnitus animals and to a much less extent in exposed-no tinnitus animals (49). Although this shift to enhancement is less pronounced in exposed no tinnitus animals, the cannabinoids might be able to increase this enhancement effect to the level comparable to that in exposed tinnitus animals. However, cannabinoids might not be able to shift the suppression-dominant effect to enhancement in sham animals. Having said this, it must be appreciated that the drug administration in this study was systemic, and therefore, the actions of THC and CBD cannot be attributed solely to the cochlear nucleus or even the central auditory system; in fact, the effects of these cannabinoids on any area of the CNS, including the limbic system that projects to the central auditory system, could conceivably have contributed to the observed effects on tinnitus-related behavior. The other issue that must be noted is that although delta-9-THC is a partial agonist at CB1 receptors, CBD can act as a partial CB1 antagonist (50), and it is impossible to know the net effect of these two drugs, even in the cochlear nucleus. Furthermore, previous studies have demonstrated that acute and chronic dose regimens with cannabinoid drugs can have quite different effects. For example, Okine et al. (51) reported that chronic pre-treatment with URB597, and inhibitor of fatty acid amide hydrolase, a key enzyme in the metabolism of the endocannabinoid, anandamide, had no effect on inflammatory pain behavior in rats, whereas a single dose significantly reduced it. It is therefore quite conceivable that we might have observed different results with acute dosing of delta-9-THC/CBD.
In addition, endocannabinoids in the central nucleus of the amygdala have been implicated in short-term adaptation of the conditioned fear response, and the CB1 receptor antagonist AM251 increased the fear response (52). Because systemic injections were used to deliver the drugs in this study, it could be argued that changes in the frequency discrimination curve might not reflect the perception of tinnitus but rather changes in the fear response through the drugs’ effects on the amygdala. However, if this was the case, cannabinoids would facilitate the adaptation of the conditioned fear response and result in an upward shift of the curve rather than the downward shift observed in the exposed no tinnitus animals. A close inspection of the curves did reveal a slight upward shift of the curve in the sham-drug animals in response to 20 kHz tones during the drug administration and in response to 20 and 32 kHz tones after drug administration, which suggests that adaptation of the conditioned fear response might have occurred in our animals. However, this adaptation was not enough to affect the greater lick suppression in animals with tinnitus.
Although Cannabis is used by some tinnitus sufferers to relieve their condition, our results, consistent with our previous study using the salicylate model (17), suggest that cannabinoids, such as delta-9-THC and CBD, may actually aggravate tinnitus (53). This might be predicted from the work of Zhao et al. (16), which suggested that the net effect of activation of CB1 receptors in the DCN might be to increase the excitation of fusiform cells, thus exacerbating neuronal hyperactivity.
Conflict of Interest Statement
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
This study was supported by a Jean Cathie Bequest Fund, administered by the Auckland Medical Research Foundation, New Zealand.